Analysis of Insulin Analogs and the Strategy of Their Further Development

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ISSN 0006-2979, Biochemistry (Moscow), 2018, Vol. 83, Suppl. 1, pp. S146-S162. © Pleiades Publishing, Ltd., 2018. Original Russian Text © O. M. Selivanova, S. Yu. Grishin, A. V. Glyakina, A. S. Sadgyan, N. I. Ushakova, O. V. Galzitskaya, 2018, published in Uspekhi Biologicheskoi Khimii, 2018, Vol. 58, pp. 313-346. REVIEW

O. M. Selivanova1, S. Yu. Grishin1,2, A. V. Glyakina1,3, A. S. Sadgyan4, N. I. Ushakova4, and O. V. Galzitskaya1*

1Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; E-mail: [email protected] 2Lomonosov Moscow State University, 119991 Moscow, Russia 3Institute of Mathematical Problems of Biology, Keldysh Institute of Applied Mathematics, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia 4Joint-Stock Scientific Production Association Bioran, 119261 Moscow, Russia Received June 5, 2017 Revision received July 9, 2017

Abstract—We analyzed the structural properties of the peptide hormone insulin and described the mechanism of its physiological action, as well as effects of insulin in type 1 and 2 diabetes. Recently published data on the development of novel insulin preparations based on combining molecular design and genetic engineering approaches are presented. New strategies for creation of long-acting insulin analogs, the mechanisms of functioning of these analogs and their structure are dis- cussed. Side effects of insulin preparations are described, including amyloidogenesis and possible mitogenic effect. The pathways for development of novel insulin analogs are outlined with regard to the current requirements for therapeutic preparations due to the wider occurrence of diabetes of both types.

DOI: 10.1134/S0006297918140122

Keywords: insulin analogs, diabetes, hyperglycemia, hypoglycemia, glycemic control, insulin fibrils

Insulin is a peptide hormone (51 a.a.) produced by Insulin is produced in response to the rise of glucose the β-cells of the pancreatic Langerhans islets. An insulin blood concentration; it binds to the insulin receptor (IR) monomer consists of two polypeptide chains: A (21 a.a.) and activates glucose transporters, mainly Glut4, in fat and B (30 a.a.) connected via two disulfide bridges tissues and cardiac and skeletal muscles. Mobilized formed by cysteine residues at positions A7–B7 and transporters are recruited from the intracellular compart- A20–B19. The third intrasubunit S–S bond is formed ments to the plasma membrane, where they facilitate glu- between the residues A6 and A11. Although insulin was cose transport into the cell [2]. If insulin production is discovered at the beginning of the XX century [1], the disrupted, the blood concentration of glucose increases studies of its functions have remained relevant up to pres- (chronic hyperglycemia), which is the basic diagnostic ent time. symptom of type 1 diabetes [3]. Type 2 diabetes develops Insulin regulates a number of metabolic processes, when the IR signaling is disturbed, even if the hormone is such as biosynthesis of proteins, fats, and nucleic acids, produced in sufficient amounts; it is characterized by the as well as cell growth and differentiation. However, its key decreased tissue sensitivity to insulin (insulin resistance) function is regulation of glucose uptake by the cells. [4-6]. Diabetes is classified as a group of metabolic diseases Abbreviations: αCT, C-terminal domain of insulin receptor α- characterized by hyperglycemia caused by defects in the subunit; BMI, body mass index; ER, endoplasmic reticulum; insulin secretion or insulin action [7]. Both reduced cell IR, insulin receptor; IGF-1R, type 1 insulin-like growth factor sensitivity to insulin and inadequate insulin levels in the receptor; IRA, insulin receptor isoform A; IRB, insulin receptor isoform B; L1, leucine-rich repeat domain of the insulin type 2 and 1 diabetes lead to development of such com- receptor α-subunit; NPH, neutral protamine Hagedorn; PEG, plications, as vascular diseases, in particular coronary polyethylene glycol; ThT, thioflavin T. heart disease, cerebrovascular disorders, retinopathy, * To whom correspondence should be addressed. nephropathy, and neuropathy [8]. Impairments in insulin

S146 ANALYSIS OF INSULIN ANALOGS S147 secretion and functioning can be found in the same erences of patients should be taken into account during patient, which makes revealing the major cause of hyper- the therapy [16]. glycemia rather problematic. The immune response of an The limitations in the use of insulin are related to the organism to its own β-cells in the pancreas can be diag- necessity of administration of exact doses several times a nosed from the presence of autoantibodies against day in order to maintain physiological levels of glucose in insulin, Langerhans islet cells, tyrosine phosphatases IA2 the blood. Besides, the hormone has a narrow therapeu- and IA2β, and glutamate decarboxylase GAD 65. An tic window associated with the risk of hyperglycemia; it increased risk of type 1 diabetes development can be can also cause weight gain that might be dangerous in revealed by molecular genetic analysis of the HLA–DQB1 patients with a high body mass index (BMI) [17, 18]. gene alleles [9, 10]. As a rule, insulin secretion at the last The above problems that are associated with the stage of diabetes is insignificant or lacking at all, which is properties of native human insulin have promoted the indicated by low blood plasma levels of the C-peptide. development of its analogs (Fig. 1). Patients with type 1 diabetes become dependent on Insulin analogs are synthetically modified molecules insulin administration. that, due to faster or more prolonged action, allow better Only 5-10% diabetes patients have type 1 diabetes, metabolic control of the blood glucose levels in diabetes while most patients have type 2 diabetes [7]. Although no [20]. By the present time, different preparations for dia- autoimmune destruction of cells takes place in type 2 diabetes therapy have been created that include insulin betes, this type of diabetes can be caused by various other analogs and mixtures. factors. The precise mechanisms of type 2 diabetes are Herein, we review existing insulin analogs. There are extensively studied [11-15]. Most cases of insulin usage two main strategies for their development: creation of are associated with type 2 diabetes, because it has a high- bolus (short-acting) and basal (long-acting) insulin er occurrence. preparations. These types of insulin analogs can be com- The efficiency of insulin in diabetes treatment that is bined to normalize glucose blood levels and to provide the related to the ability of this hormone to decrease the delivery of the preparations in a form convenient for blood glucose levels has been demonstrated during patients. decades of insulin use in medical practice. However, at The use of insulin as a drug has started almost imme- present, novel approaches to the diabetes therapy remain diately after its discovery [1]. Since then, numerous bio- a topical problem, because the number of disease cases chemical and biomedical studies have been conducted in continues to rise. Moreover, individual features and pref- order to further improve insulin preparations.

Detemir

Glargin

Glargin Chain A Aspart

Lispro

Chain B

Fig. 1. Structure of insulin and its analogs (modified from [19]).

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 S148 SELIVANOVA et al. BIOSYNTHESIS AND SECRETION OF INSULIN erol. Inositol 1,4,5-trisphosphate is a ligand for the ER receptor proteins responsible for the release of intracellu- Insulin biosynthesis starts with the translation of lar calcium that results in an increased cytoplasmic Ca2+ mRNA for its precursor, preproinsulin. The precursor concentration. Finally, calcium ions stimulate insulin (110 a.a.) is encoded by the INS gene, a single copy of release from the secretory granules. Besides glucose, which is located in the short shoulder of chromosome 11 of other molecules that mediate insulin secretion have been the human genome [21]. Preproinsulin is synthesized only found: nicotinamide adenine dinucleotide phosphate on polyribosomes associated with the endoplasmic reticu- (NADP), glutamate and malonyl-CoA [25, 26], glycerol- lum (ER). The signal peptide (24 a.a.) of preproinsulin is 3-phosphate [27], and fatty acids [28, 29]. Insulin is cleaved off by the signal peptidase as the polypeptide is released from the cells through exocytosis: a mature translocated into the ER lumen [22] forming proinsulin. secretory granule fuses with the plasma membrane and In the ER, proinsulin folds into correct conformation with releases its content to the extracellular space. the formation of three disulfide bonds (B7–A7, B19–A20, A6–A11). Conversion of proinsulin into monomer insulin and C-peptide happens after proinsulin transport from the STRUCTURAL PROPERTIES AND SURFACE ER to the Golgi apparatus, where proinsulin is cleaved by CONTACTS IN THE INSULIN MOLECULE. peptidases in secretory vesicles, during which the C-pep- BINDING TO THE RECEPTOR tide (of 31 a.a.) located between fragments B and A is excised from the molecule. Insulin is then stored as a hexa- Immediately after insulin hexamers are secreted mer coordinated by two Zn2+ ions in β-cells of the from the β-cells and diffuse in the blood, a combination Langerhans islets in the pancreas [23, 24]. of electrostatic repulsion and insulin concentration gradi- An increase in the glucose blood level acts as a signal ent promotes dissociation of the hexamers into dimers for insulin secretion. As a rule, the process starts when the and monomers (only monomers exhibit biological activi- insulin-independent carrier protein Glut2 binds glucose ty). Therefore, hexamers are the storage form of insulin, molecules and transports them into β-cells of the while monomers are the biologically active form of this Langerhans islets. In β-cells, glucose is phosphorylated hormone (Fig. 2). by the enzyme glucokinase and eventually converted to Insulin monomer consists of chains A and B con- pyruvate via glycolysis. Pyruvate is oxidized in the tricar- nected by disulfide bonds. The secondary structure of boxylic acid cycle with the formation of ATP. An increase chain A contains two antiparallel α-helices formed in the ATP/ADP ratio leads to the closing of ATP- between residues A2-A8 and A13-A19 connected by the dependent potassium channels and depolarization of the fragment A9-A12. The secondary structure of chain B plasma membrane. As a result, voltage-dependent potas- contains β-strands and an α-helix. Amino acid residues sium channels open and potassium enters the cells. High B1-B5 form an elongated region; the central α-helix is intracellular potassium concentrations stimulate phos- preceded by a sharp 1→4 turn due to the presence of pholipase C that cleaves phosphatidylinositol 4,5-bispho- glycine residue B8. This explains the tight packaging of sphate into inositol 1,4,5-trisphosphate and diacylglyc- the α-helix by the 310 type via hydrogen bonds formed by

a b c

Chain A

Chain B

Fig. 2. X-ray structure of human recombinant insulin at 0.92 Å resolution: a) monomer; b) dimer; c) hexamer coordinated by two Zn2+ ions (PDB 5E7W, visualization in YASARA).

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 ANALYSIS OF INSULIN ANALOGS S149 B7, B8, and B9 [30]. Starting from Ser at position B9, stitution for Phe in position B25 (Chicago insulin), Ser hydrogen bonds promote formation of the helical struc- substitution for Phe in position B24 (Los Angeles ture of the 1→5 type, and the α-helix is maintained insulin), and Leu substitution for Val in position A3 through B19. Then, the 1→4 β-turn is formed by the (Wakayama insulin) suffer from type 2 diabetes [42-46]. hydrogen bonds between B19 carbonyl oxygen and B22 Insulin binding to the receptor was reduced by 30%, amide hydrogen and between B20 carbonyl oxygen and when the N-terminal nitrogen of chain A was acetylated, B23 amide hydrogen. Residues B23-B30 form the β- which shows the importance of free positively charged strand. PheB24 and TyrB26 interact with LeuB11 and amine group in A1 [33]. Removal of Gly in A1 decreased LeuB15 of the chain B α-helix. Cysteine residues at each insulin affinity to the receptor by 15%, demonstrating end of the chain B helical segment stabilize the native that the salt bridge formed by GlyA1 and the C-terminus structure of insulin by forming disulfide bonds with chain of chain B is essential for the correct positioning of the A (A7–B7 and A20–B19). peptide [47]. It should be noted that the overall confor- Chain B can have two different conformations in a mation might be more important than the presence of crystal [30]. In the T conformation, the central α-helix certain amino acid residues. For example, the replace- runs from B9 through B19, while the N-terminus (B1- ment of GlyA1 with L-amino acids (Ala, Val, Leu, Pro, B8) is unfolded. In the R conformation, the α-helix is Trp, Lys, or Glu) reduces insulin binding to the receptor continuous (B1-B19). The transition from the T confor- by 2-20%, while replacement of the same GlyA1 with D- mation to the R conformation was studied in a solution of amino acids (D-Phe, D-Leu, D-Trp, D-Ala, D-Lys, or insulin hexamers; the changes in the content of α-helices D-Glu) does not affect the biological activity of insulin were registered using circular dichroism [31] and 2D- analogs [48-51]. Mutations impairing the folding of the NMR [32, 33] methods. It was shown that the T6→R6 chain A α-helix (as determined by a decreased molar transition occurs in the presence of phenols and some ellipticity of circular dichroism) also result in reduced other cyclic alcohols [34] including cyclohexanol [35]. biological activity [52]. Addition of phenol ligands induces a conformational The structure of chain B is especially well studied. It transition of the N-terminus in chain B not only in native was demonstrated that the first four residues of chain B do insulin, but also in its analog insulin lispro (LysB28, not affect the activity of insulin binding to the receptor, ProB29) [36]. The monomeric forms of insulin acquire while the removal of HisB5 leads to its considerable the R-like conformation in a solution. The secondary reduction [52, 53]. LeuB6 is of critical importance structure of both A and B chains determines the affinity of because deletion of this amino acid residue results in 99% insulin to its receptor, so it was suggested that the T→R loss of affinity to the IR [54]. Substitution of His with Asp transition is important for the binding and activation of in B10 in a synthetic insulin analog causes a 500% IRs. increase in its affinity to the IR [55]. However, because IR is a homodimer, each subunit of which consists of HisB10 is essential for insulin biosynthesis, its substitu- two (α and β) polypeptide chains. The extracellular α- tion with AspB10 prevents insulin formation from proin- subunit binds insulin, while the transmembrane β-sub- sulin, which leads to an elevated concentration of the lat- unit contains a tyrosine kinase intracellular domain [37]. ter [56, 57]. The C-terminal fragment of chain B contains Insulin monomer binds to the site formed by two α-sub- evolutionary conserved residues involved in the receptor units. This site includes the L1 (leucine-rich repeat binding (GlyB23, PheB24, PheB25, and TyrB26). In par- domain) of one α-subunit and the carboxyterminal ticular, PheB24 forms hydrogen bonds critical for dimer- (αCT) domain of the other subunit. The binding of ization. The conformation of PheB25 is different in the insulin leads to the repositioning of the helix in the αCT two molecules forming the dimer, i.e., this residue is domain on the L1 surface in such a way that it interacts, important for the generation of insulin native structure on the one hand, with insulin and, and on the other hand, [47]. Substitution of PheB24 with methionine or cyclo- with L1. The C-terminus of chain B shifts relatively to the hexylalanine decreases the time of disassembling of other parts of the molecule after binding with αCT [38, insulin analog hexamers [40]. 39]. PheB24 plays the role of an anchor in the nonpolar According to the crystallography data, the C-termi- pocket formed by the L1 surface, αCT, and the central α- nus of chain B is essential for the dimer formation. The helix of chain B [40]. It is believed that only three regions amino acids at positions B8, B9, B12, B13, B16, B23-B28 close to the insulin molecule surface are responsible for form the contacts between the insulin molecules. the interaction with the IR because the residues in all the Essential amino acids, whose substitution results in a sig- three regions are evolutionary conserved: the N-terminus nificant decrease in the capacity of insulin to form of chain A (GlyA1-IleA2-ValA3-GluA4), the C-terminus dimers, were identified by mutagenesis [58]. Sequential of chain A (TyrA19-CysA20-AsnA21), and the C-termi- truncation of the insulin molecule from the C-terminus nus of chain B (GlyB23-PheB24-PheB25-TyrB26) [41]. of chain B showed that ProB28 is critical for the insulin Peptides with mutations in these regions have lower affin- self-association in a solution. Removal of ProB28, its ity (1-5% of the normal) to the IR. Patients with Leu sub- substitution, or, especially, inversion of amino acids in

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 S150 SELIVANOVA et al. positions B28 and B29 (Lys-Pro) almost completely elim- injected subcutaneously, insulin hexamers should dissoci- inate the capacity of insulin for the dimer formation, but ate into monomers at the site of injection prior to getting does not affect its ability to form hexamers [59]. It should into the bloodstream. This delay in the onset of insulin be noted that in all commercial pharmaceutical prepara- action depends on the injection site and causes variability tions, insulin is in hexamer form [60]. in the insulin action profile and discrepancy between the time of insulin administration and food intake. Patients are required to take an insulin dose 15-30 min before the INSULIN ANALOGS meals, which is problematic in everyday life [64]. To improve the control of glucose blood levels, insulin Elucidation of the insulin structure and identifica- preparations with different time courses of action have tion of most important regions responsible for the forma- been developed (Table 1). tion of dimers and hexamers and binding to the IR signif- Weakening of contacts between the monomers in the icantly promoted the development of insulin analogs. insulin dimer allowed to obtain short/rapid-acting Because insulin is active in its monomeric form, creation analogs (bolus insulins), such as insulin lispro of insulin analogs with a modified structure should lead to (Humalog®), insulin aspart (NovoRapid®), and insulin prolongation of their action and a change in the onset of glulisine (Apidra®). The onset of action of these analogs insulin effects in the body. is within 5-15 min after subcutaneous administration; the Basal and bolus analogs of insulin. The aim of insulin peak of action is within 1-2 h; and the duration of action replacement therapy is the maximal imitation of physio- does not exceed 4 h. Therefore, these preparations are logic insulin secretion, when small amounts of insulin are designed for effective control of hyperglycemia immedi- discharged continuously (the so-called basal or back- ately after meals. ground secretion). Rapid secretion of insulin by β-cells in The slowing down the dissociation of insulin hexam- response to food intake is called bolus secretion [61]. ers into monomers led to the development of long/ultra- Insulin therapy is recognized as an efficient way to pre- long-acting preparations, e.g., insulin glargin (Lantus®) vent macro- and microvascular complications in patients and insulin detemir (Levemir®). The unique property of suffering from type 1 diabetes. To minimize degenerative these preparations is the absence of peak increase in the changes in organs, patients with diabetes should maintain insulin blood level and the ability to maintain stable the glucose blood levels that would correspond to normal insulin concentration for 24 h. In other words, these insulin secretion [62]. At the same time, regular adminis- insulin preparations almost completely supplement the tration of insulin causes a number of adverse side effects, basal secretion of insulin typical of healthy subjects. The such as an increased risk of pronounced hypoglycemia, low absorption rate of long-acting insulin analogs allows weight gain, and changes in the action profile of insulin administering them once a day. The onset of their action [16, 63]. When insulin is injected intravenously, its hexa- is on average 1 h after subcutaneous injection, and the mers almost immediately dissociate into monomers; the duration of action may reach 24 h (Fig. 3). monomers interact with IRs in the target tissues and Therefore, combined administration of ultrashort- decrease the glucose levels almost instantaneously. When and long-acting insulin analogs might completely imitate

Table 1. Characteristics of insulin preparations [65-68]

Bolus preparations

Short-acting insulin Analogs of rapid-acting insulin

Duration of action is 6 h or more; Rapidly absorbed and act for 3-4 h; onset of action is 30 min after administration; act faster than short-acting insulin and are taken during meals frequently acts longer than required, therefore, some patients to restrain the postprandial increase in blood glucose run the risk of hypoglycemia

Basal preparations

Neutral protamine Hagedorn (NPH) Analogs of long-acting insulin

Duration of action is 8-12 h or more; Duration of action is 24 h; taken twice a day; slowly absorbed; might become turbid and should be resuspended by shaking the taken once (sometimes twice) a day bottle/pen; some patients can be more prone to hypoglycemia as compared to administration of long-acting insulin

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 ANALYSIS OF INSULIN ANALOGS S151

Aspart, lispro (4-6 h)

Regular insulin (6-10 h) NPH (12-20 h) Detemir (18-24 h) Glargin (20-24 h) Serum insulin level, mU/liter

Time, h

Fig. 3. Pharmacokinetic profiles of insulin analogs (modified from [19]). the two-phase physiological secretion of insulin in children with diabetes and requiring insulin therapy healthy organisms. The use of these preparations is asso- receive genetically engineered analogs of human insulin ciated with a lower risk of hypoglycemia, which is the as the most efficient and safe preparations. most hazardous complication of insulin therapy. The most important feature of the Russian insulin Humulin® was the first insulin preparation pro- market is that for more than twenty years it has been con- duced with the use of recombinant DNA technology [69]. trolled by foreign manufacturers with three pharmaceuti- Different insulins have been designed since then: neutral cal companies being the leaders in this field: Novo protamine Hagedorn (NPH), rapid-acting analogs, basal Nordisk, Eli Lilly, and Sanofi [71] (Table 2). analogs, and premixed insulin analogs from such compa- Up to the present, the criterion of the Russian nies as Eli Lilly (USA), Novo Nordisk (Denmark), and Ministry of Industrial Trade for a local preparation has Sanofi (France) [70]. been the recognition of the packing stage, which is suffi- Production and application of insulin analogs. The cient for a foreign preparation to get a license for its distri- first medical insulin preparations had been produced bution in Russia. The most popular imported insulin from animal pancreatic extracts. However, the amino acid preparations in Russia are Actrapid HM and Protaphane sequences of bovine and porcine insulins differ from that HM (Novo Nordisk) and Humulin regular and Humulin of human insulin (in the porcine insulin in the B chain, NPH (Eli Lilly). These preparations are analogs of Insuran instead of the threonine in the B30 position, alanine is P and Insuran NPH (Shemyakin and Ovchinnikov found; in the bovine insulin in addition to this substitu- Institute of Bioorganic Chemistry, Russian Academy of tion, in the A chain, instead of the threonine in the A8 Sciences), respectively. position, alanine is found, and in the A10 position, In 2014, the patent terms for Levemir® and instead of isoleucine, valine is found), therefore animal NovoLog® (active components are detemir and aspart, insulin preparations caused adverse side effects in humans respectively) from Novo Nordisk and for Humalog® (e.g., allergy). Besides, due to the limited amount of (insulin lispro) from Eli Lilly expired, which allowed source materials, animal insulins could not satisfy the other companies to start manufacturing analogous insulin requirements for the insulin preparations. products, the so-called biosimilars (Table 3). The invention of recombinant DNA technologies The characteristics of biosimilars are analogous to allowed for the first time the large-scale production of those of the original preparations. To get a permission recombinant insulin identical to the human insulin as from the regulating authorities to produce biosimilars, the well as production of its analogs with designed properties manufacturing company should prove that the biosimilar and predetermined duration of action. is safe, efficient, and similar to the original drug and pro- Production of insulins with different duration of vide identification of the patent status of the original bio- action is of great importance, because combination of pharmaceutical preparation. A biosimilar may not be such insulins in one preparation makes it possible for dia- identical to the original preparation, but should exhibit betes patients to administer the drug directly before the similar therapeutic action. The terms of patent defense meal and to conveniently decrease the number of injec- and exclusive access to the market should be terminated tions. in the area of patent action. In the developed countries and Europe, almost all Biosimilars include commercially available analogs diabetes patients in need of insulin therapy are treated of glargine, such as Basalog® (Biocon Ltd., Bangalore, with genetic engineered insulin analogs. In Russia, 100% India) and Basalin® (Gan & Lee, Beijing, China), and

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 S152 SELIVANOVA et al.

Table 2. Percentage of producers of insulin and its analogs sold in the Russian Federation in 2015-2016 according to the Klifar-Goszakupki Data Analysis System

Producers of insulin and its analogs 2015 First six months of 2016

Novo Nordisk (produced by Novo Nordisk, Denmark) 37.1% 40.1% Sanofi (produced by Sanofi, Germany) 35.9% 34.4% Eli Lilly (produced by Eli Lilly, USA) 18.2% 16.6% Pharmstandard (produced by Sanofi) 3.9% 3.7% Medsintez plant (presumably produced by Chinese pharmaceutical industry, China) 2.5% 2.3% P-Pharm (packing of finished dosage form (FDF) produced by Eli Lilly, USA) 1.2% 0.7% Glaxosmithkline (British distributors of FDF produced by Bioton S.A., Poland) 0.5% 0.2% Geropharm (complete cycle, its own preparation; Russian Federation) 0.4% 1.9% Marvel Lifesciences (India) 0.2% 0.1% Wockhardt (the company plans to organize the location for the packing stage via PharmEco, India) 0.2% 0.1% Total: Top three 91.3% 91% Total: Top ten 100% 100%

Table 3. Main current insulin biosimilars [16, 72]

Developer Products Test phase

Eli Lilly (USA) LY2963016; presented in EU (July 2013) innovative basal preparation LY2605541 phase III

Sanofi (France) U-300 (Lantus); phase III Lixisenatide + Lantus phase I/II

Novo Nordisk (Denmark) Tresiba (degludec); tested in USA, registered in EU Ryzodeq (Tresiba + Novolog);

IDegLira (liraglutide + degludec); presented basal peroral insulin (pills); phase I product diversification, phase III FIAsp (Novorapid)

Biocon (India) biosimilars (based on Lantus, Humalog, Novolog) phase I

Biocon Bristol-Myers Squibb IN-105, prandial peroral insulin phase I/II (EU) Research Center (India)

Biodel (USA) Humalog biosimilar; phase II Lantus biosimilar phase I (USA)

MannKind Corporation (USA) Afrezza (ultrashort-acting inhaled insulin) phase III (USA)

premixed insulin compositions Gensulin® (Bioton S.A., efficiency of genetically engineered human insulin prepa- Warsaw, Poland), Insugen® (Biocon Ltd, Bangalore, rations registered in the Russian Federation does not India), Wosulin® (Wockhardt Ltd, Mumbai, India), and require the proof that these preparations have character- Biosulin® (MJ Biopharm Pvt Ltd, Mumbai, India) [72]. istics analogous to the original ones. So strictly speaking, Since 2000, the number of genetically engineered these preparations are not bioanalogs. At the same time, preparations of human insulin that have been developed they cannot be recognized as original medical prepara- and registered in the Russian Federation has grown essen- tions because their preregistration studies have been sig- tially. The procedure for the confirmation of safety and nificantly reduced.

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 ANALYSIS OF INSULIN ANALOGS S153 To confirm that novel preparations of genetically quadriceps muscles proceeds slower than in the abdomi- engineered human insulin and its analogs represent nal cavity and results in prolongation of the action of both bioanalogs of the original preparations, it is necessary to regular insulin and insulin lispro, as evidenced by phar- follow earlier developed recommendations [73-75], as macokinetic and pharmacodynamic data [66]. well as use the new ones. The other essential component of the glucose level In addition, the emerging willingness to comply with control in insulin-dependent diabetes is the use of long- the European standards on the confirmation of the acting insulin analogs to substitute the basal insulin in bioanalogous nature of medical preparations will allow order to reduce the risk of nocturnal hypoglycemia that the development of scientific and methodical procedures, could be caused by administration of human insulin as well as the program for preclinical and clinical studies preparations [85, 86]. Neutral protamine Hagedorn of genetically engineered bioanalogs of human insulin (NPH), as well as insulins lente and ultra-lente [67, 87], and its analogs, and will lead to new approaches to the were the first preparations used to maintain the basal hor- evaluation of the results of these studies in order to esti- mone level. NPH is obtained by adding protamine and mate the benefits vs. risks of the developed drugs. Zn2+ to insulin. This procedure results in the formation of Strategies and results of insulin analog design. From heterogeneous suspensions, which slightly increases the the theoretical point of view, the differences in the action duration of the insulin action. However, the reproducibil- profiles of insulin analogs make it possible for the patients ity of the action profiles of such insulin preparations with type 1 diabetes to dynamically control glucose blood remains low, which in part might be related to the neces- levels in order to avoid hyper- and hypoglycemia. sity to resuspend insulin in a vial before administration. However, such control requires imitation of the activity of The differences in the duration of action of these insulin healthy pancreas, i.e., maintenance of basal insulin levels preparations can be explained by the stochastic release of with peak increases in the hormone concentration during hexamers from the aggregates [88]. meals [76, 77]. The strategy based on diminishing the solubility of The first designed insulin analog was rapid-acting injected preparation at the injection site has proven to be insulin lispro that was released on the market in 1996. As more effective for designing basal insulin preparations. described above, the action of insulin lispro is based on For example, modifications in insulin glargin led to the the weakening of self-association of insulin molecules by shift in the molecule isoelectric point in order to signifi- inversion of amino acid residues ProB28 and LysB29 [78]. cantly reduce its solubility at physiological pH, which The Pro–Lys inversion changes the local conformation of makes injected insulin glargin much less soluble [68, 89, the chain B C-terminus by eliminating two critical 90]. The increase in the isoelectric point was achieved by hydrophobic interactions, which results in the weakening introducing two Arg residues at positions B31 and B32; of the two terminal hydrogen bonds in the β-sheet and additionally, Gly was introduced at A21 to provide the insulin dimer destabilization [79]. Phenol ligands induce chemical stability of the resulting preparation. It was conformational changes by binding to specific sites on the found that both human insulin and its analogs were insulin hexamer and promoting transition of eight N-ter- equally efficient in the glucose level control, but the minal residues in chain B from the elongated conforma- analogs counteracted the effects of hypoglycemia better, tion to the α-helix [80]. Another classical short- and especially at night. However, insulin glargin was found to ultrashort insulin analogs are aspart and glulisine, which cause weight gain in diabetes patients [91]. The strategy dissociate into dimers and monomers faster than regular used for developing glargin has been rejected by now human insulin. Therefore, they are more rapidly because of the inflammatory response occurring at the absorbed, but have a shorter duration of action [81-83]. site of injection [16]. During the first hour after administration, the phar- An alternative approach for developing long-acting macokinetic profile of the lispro plasma concentration is insulin analogs is acetylation of LysB29 with a fatty acid two times higher than that of human insulin. Insulin residue, which leads to the reversible hormone binding to lispro also reaches its maximal concentration two times the blood serum albumin and prolongation of its action. faster than regular insulin. Three to four hours after injec- This strategy was used to develop insulins detemir [92] tion, the concentration of rapid-acting insulins drops to and degludec [93, 94]. Insulin degludec (Tresiba, Novo 20% of the maximum, while regular insulin is still Nordisk) is human insulin in which LysB29 is modified absorbed at the injection site [84]. Insulin lispro more with a palmitic acid residue and ThrB30 is deleted (de- efficiently lowers the glucose level 0-4 h after the meal, B30). The medical preparation of insulin degludec con- which makes it possible to administer it within 15 min tains phenol and zinc; in the presence of these compo- after beginning of the meal. Unlike regular human nents, it exists in a stable dihexameric form, where the insulin, the time for the onset of the insulin lispro effect fatty acids are positioned between the hexamers and inter- depends less on the site of injection. However, it is rec- act with PheB1 residues in each chain [95]. The duration ommended to inject rapid-acting insulin analogs into the of action of insulin degludec is determined mainly by its abdominal wall, because absorption in the deltoid and oligomeric structure, whereas the binding to the serum

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 S154 SELIVANOVA et al. albumin provides for the amortization effect and decreas- in the same way as bulk amino acids do it in truncated es variability of the action profile due to more uniform insulin analogs N-MePheB26 and N-MeTyrB26 [103]. uptake into the bloodstream [93]. The clinical assessment The novel strategies for the design of basal insulin of insulin degludec demonstrated that decreased levels of preparations include primarily three directions: (i) insulin glucose are maintained for up to 42 h with simultaneous binding to glucose-responsive polymers for controlled reduction of the episodes of nocturnal hypoglycemia, as release of the hormone [62], (ii) rearrangement of insulin compared to the use of insulin glargin [96]. hexamers [104], and (iii) pH-dependent binding of zinc Another trend in the development of basal insulins is ions by created His-X3-His sites (“histidine staples”) on creation of hepatoselective analogs with a higher poten- the hexamer surface [105, 106]. tial for attenuation of glucose levels and lower risk of Hence, insulin analogs can be differentiated by the hypoglycemia and body weight growth than those of reg- profile of their action in the following way: ular insulin [97]. For example, insulin PEGLispro (Eli 1) short- and ultrashort acting analogs that imitate Lilly) was obtained by attaching polyethylene glycol the postprandial insulin secretion; (PEG) to the insulin molecule [98]. This modification 2) long-acting (peakless) analogs designed to imitate increases the hydrodynamic radius of the molecule, a stable (basal) profile of insulin secretion by healthy pan- which, on one hand, allows insulin PEGLispro creas. monomers to penetrate via liver capillaries but, on the At present, the basal-bolus therapy is used because it other hand, prevents penetration via capillaries of other is more flexible than other regimes [107-110]. As to the tissues, such as fat tissue or muscles. According to phar- intermediate-acting insulins, the widely used long-acting macokinetic studies, the half-time of insulin PEGLispro insulin preparations have made them irrelevant. elimination was prolonged to 2-3 days. No difference was observed in the frequency of hypoglycemia in patients taking insulins PEGLispro and glargine, although the fre- FIBRILLATION OF INSULIN PREPARATIONS quency of nocturnal hypoglycemia in the first group was lower [99]. Regrettably, these studies were terminated Insulin possesses a high propensity for aggregation because clinical trials showed that insulin PEGLispro and formation of fibrils (fibrillation). This disposition for impairs liver functions; in particular, higher levels of polymerization, as well as the possibility to obtain repro- enzymes alanine aminotransferase and aspartate amino- ducible kinetic data, explains the attention that insulin transferase were observed [100]. has received as a model object for studying fibril forma- An important direction in the development of insulin tion. When searching for different insulin monomeric preparations is design of analogs specific to particular isoforms, researchers have also investigated insulin capacity forms of the IR, since it was suggested that some insulin for aggregation and fibrils formation. analogs can promote activation of the IGF-1 receptor, Fibrillation is undesirable in insulin therapy. Regular which might result in the tumor growth stimulation [101, non-mutant insulin used for injections is prone to fibril- –11 102]. Insulin binds with a high affinity (Kd ∼ 5·10 M) to lation under the action of various external factors. Insulin the IR and with lower (but still noticeable) affinity (Kd ∼ preparations often spontaneously polymerize during stor- 10–8 M) – to IGF-1R (type 1 insulin-like growth factor age and transportation. There is also a problem of insulin- receptor) [33]. The functions and tissue expression of the derived amyloidosis upon frequent insulin injections. IR isoforms IRA and IRB differ. The physiological meta- Aggregation of insulin into insoluble fibrils may bolic effect of insulin is mediated by IRB, while excessive cause numerous complications, such as an attenuated hormone biding to IRA and IGF-1R might cause pro- therapeutic effect of insulin, clogging of insulin delivery nounced mitogenic effect. In vitro models demonstrated systems, or activation of immune response. Fibrillation that short-acting insulin analogs bind to IRA, IRB, and might affect the glucose level control; besides, it raises the IGF-1R, similarly to insulin. On the contrary, long-act- cost of treatment and decreases its efficiency [111]. ing analogs, such as insulins glargin and detemir, prefer- Identification of regions in the insulin molecule, ably bind to IGF-1R and to a lesser extent – to IR. which are responsible for the aggregation and stability, Therefore, insulin analogs predominantly activate the might promote the development of novel insulin analogs ERK kinase signaling pathway, while regular insulin acti- for better regulation of glucose blood levels and reduce vates AKT kinase [20]. the propensity of insulin for fibril formation by decreasing The receptor-binding activity of the hormone can be the probability of its self-association [112]. The early enhanced by the following modifications: removal of stages of fibrillation are of particular interest [113]. residues B27-B30 at the C-end of chain B together with It is believed that the key role in the growth of insulin amidation of the C-terminal residue in position B26 and fibrils belongs to the conformational shift of the chain B inclusion of N-methylated amino acid or D-enantiomer. C-terminus that exposes a domain containing amino acid However, methylation of TyrB26 or its substitution with N- residues A2, A3, B11, and B15. In its turn, this protrud- methylated alanine decreases the receptor-binding activity ing domain interacts with another hydrophobic domain

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 ANALYSIS OF INSULIN ANALOGS S155 containing residues A13, B6, B14, B17, and B18 [114]. Substitution of ThrA8 with His slows down insulin fibril- a lation [115]. Disulfide bridges also play an important role in insulin fibrillation. A disruption of the A7–B7 disulfide bond induces unfolding of the insulin molecule and promotes fibrillation more than disruption of the intrachain A6–A11 disulfide bond [116]. Apparently, this is related to a low propensity for fibrillation of single-chain insulin analogs [117]. Although the kinetics of fibril formation in native insulin has been actively studied [118-120], the data on fibrillation of insulin analogs are scarce. As has been mentioned, at present there are insulin analogs with different time of action, such as rapid-acting insulins (lispro, aspart, etc.) and long-acting insulins (glargin, degludec, etc.). It has been shown that in most insulin analogs, for- 200 nm mation of fibrils occurs slower than in regular insulin b [115]. Fibrillation of rapid-acting insulin analogs has been studied in detail. Insulins lispro, aspart, and glulisine have 50 nm a longer lag-period and a lower fibrillation rate, as com- c d pared to regular insulin. However, the data on the differences between fibrillation rates of these analogs from different studies are contradictory [121, 122]. As a rule, the 10 nm most attention in the design of rapid-acting insulins is focused on amino acid residues involved in the dimer for- Fig. 4. Electron microscopy of recombinant zinc-free human mation, but not in hexamerization. Amino acid residues insulin in 20% acetic acid (pH 2.0), 140 mM NaCl after 24 h responsible for interaction with the IR are also taken into incubation at 37°C (2 mg/ml) (modified and adapted from [123]). account (Table 4). a) Observation field; b) fragments of fibrils with the smallest diameter; c and d) single fibrils under highest magnification: For our studies, we used recombinant insulin and c) packing of ring oligomers in a ring-to-ring manner, top view insulin lispro (both preparations were kindly provided by (two images); d) side view. the Bioran Scientific & Production Corporation, Russian Federation). As mentioned above, insulin lispro is a rapid-acting insulin analog and differs from the regular the recombinant insulin is about 8-10 h (2 mg/ml; 20% human insulin by the inversion of chain B amino acid acetic acid, pH 2.0, 140 mM NaCl; 37°C). Active poly- residues at positions 28 and 29 (inversion of ProB28(Lys) merization of insulin fibrils occurs after 10-12 h of incu- and LysB29(Pro)). These residues are involved in dimer- bation under the same conditions; formation of large ization of insulin molecules and at the same time do not clusters of fibrils happens after 24 h of incubation. The lag interfere with the formation of insulin hexamers. period for insulin lispro is 5 h longer than for the recom- The key research methods in studying fibrillary binant insulin (under the same conditions). However, structures are fluorescence spectroscopy (binding of according to the electron microscopy data, the morphol- thioflavin T (ThT)), electron microscopy (fibril forma- ogy of fibrils in recombinant insulin and insulin lispro is tion), and X-ray analysis (the presence of specific reflex- identical. In both cases, mature fibrils are 10 to 12 µm ions). long, and the diameter of the thinnest single fibrils is We have found that the fibrillation rates of human about 6-7 nm (Figs. 4 and 5). recombinant insulin and insulin lispro are different [124]. An increase in the incubation time resulted in a con- As shown by fluorescence spectroscopy, the lag period for siderable polymorphism of mature fibrils that interact lat-

Table 4. Amino acid residues involved in dimer and hexamer formation and binding to IR [58, 104, 114, 123]

Dimer formation Hexamer formation Binding to IR

A21, B8, B9, B12, B16, B20, B21, A13, A14, A17, B1, B2, B4, B10, B13, A1, A5, A19, A21, B12, B16, B23, B24, B25 and segment B23-B28 B14, and B17-B20 (B24 and B25 are major interacting residues)

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 S156 SELIVANOVA et al. oligomers in a fibril are either positioned in a ring-to-ring a fashion by touching their lateral sides or slightly overlap each other like dominoes. If the ring-like oligomers in a fibril are adsorbed on the Formvar film by their lateral sides, it is possible to estimate the height of a ring-like oligomer, which is approximately 3-4 nm. It should be noted that in ribbons and bundles, single fibrils are associated laterally, so that butt sides of ring-like oligomers interact with each other. Therefore, we can only see the side surfaces of oligomers, their height being ~3 nm. This height corresponds to the diameter of single fibrils in ribbons and bundles [126]. The X-ray images of fibrillar insulin show the existence of two reflexions characteristic of cross-β structure: meridional (4.8 Å) and equatorial (10.7 Å). The equatorial reflexion is very diffuse; in addition, there is also a 30 Å reflexion that has been ascribed to 200 nm the distance between laterally positioned fibrils. These b data correlate well with the earlier obtained results [127, 128]. The cross-β structure implies that a fibril consists of β-sheets positioned at a distance of 9-11 Å from each 50 nm other and parallel to the fibril axis, and the β-sheet itself is c d formed by β-strands located at a distance of 4.6-4.8 Å and positioned perpendicularly to the fibril axis [129]. Consequently, a question arises on how the 10-15-µm- 10 nm long fibrils with a diameter of 6-10 nm are formed by β- sheets positioned along the whole fibril axis. In our opin- Fig. 5. Electron microscopy images of recombinant insulin lispro ion, upon insulin fibrillation the main building block is a in 20% acetic acid (pH 2.0), 140 mM NaCl after 24 h incubation ring-like oligomer. The diffraction pattern obtained for at 37°C (2 mg/ml) (modified and adapted from [123]). a) insulin may evidence that ring-like oligomers contain Observation field; b) fragments of fibrils with the smallest diame- small β-sheets, which is the reason for a diffraction pattern ter; c and d) single fibrils under highest magnification: c) packing of ring oligomers in a ring-to-ring manner, top view (two images); characteristic of the cross-β structure. It should be noted d) side view. in this connection that as early as in 1953, Koltun et al. demonstrated using X-ray analysis that fibrils can include insulin molecules in their normal folded state [130]. Based erally with each other with the formation of ribbons of on the data obtained, we suggest that the main building different widths, twisted ribbons, and bundles of different block in the formation of insulin fibrils is a ring-like diameter. When the incubation time was prolonged even oligomer, i.e. a hexamer (Figs. 6 and 7). This model of fib- more, large clusters of insulin fibrils were formed [125, rillation might explain the polymorphism of fibrils and 126]. their breakage, branching, and surface roughness (due to According to a simplified scheme, fibrillation pro- irregular association of ring-like oligomers). It should be ceeds as follows: destabilized monomers → oligomers → also noted that hexamers involved in the fibril formation mature fibrils → fibril aggregates of different morphology. are structurally different from the zinc-coordinated hexa- As demonstrated by electron microscopy, oligomer parti- mers in the crystal. This is indirectly indicated by the fact cles are formed at the early stages of fibrillation. that no fibrillation takes place upon cross-linking of Formation of fibrils and their lengthening triggers gradual residues A1 and B29 [114]. The formed bond prevents disappearance of oligomer particles. This transition from structural rearrangement of the monomer, thereby elimi- oligomers to fibrils is the most puzzling stage of fibrilla- nating the possibility of acquiring the conformation tion. When observed under high magnification (Figs. 4c required for the fibril formation. Moreover, it also prevents and 5c), mature single fibrils appear to be formed by ring- binding of the modified insulin monomer to the IR [131]. like oligomers with a diameter coinciding with the diameter of oligomers at the initial stage of fibrillation. When The requirements for insulin replacement therapy such a fibril is placed with its ring oligomers on a support for the treatment of diabetes have promoted considerate (formvar film), it is possible to estimate the size of a ring interest in the development of insulin structural analogs. oligomer. For both insulin preparations, the outer diame- The studies abroad and in Russia have resulted in the cre- ter of ring-like oligomers (fibril diameter) is about 6-7 nm, ation of various bolus and basal insulin preparations. and the inner diameter is about 2 nm. The ring-like Nevertheless, the control of glucose blood levels without

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 ANALYSIS OF INSULIN ANALOGS S157

50 nm

10 nm d

c e a b

10 nm

50 nm

Fig. 6. The mechanism of amyloid fibril formation by human insulin and insulin lispro (modified and adapted from [123]). a) Destabilized monomers; b) ring oligomers; c) differently oriented fragments of single fibrils; d) ribbons (lateral association of fibrils); e) bundles of fibrils.

~ 2 nm and

and

A1 and B29

~ 3 nm

Fig. 7. Model of packing of three insulin hexamers (designed using the YASARA program). Dimer insulin (PDB code 5E7W) was used as the minimal structural unit. Amino acid residues GlyA1 and LysB29 important for the fibrillation are highlighted. the risk of hypoglycemia, as well as insulin delivery in a 1. The hexamer form of insulin is more stable; there- form convenient for the patients, still remain the major fore, amino acid substitutions should not involve residues strategic aims in diabetes therapy. The following should responsible for hexamer formation. be taken into account when developing new insulin 2. When substituting residues involved in insulin analogs for therapeutic purposes. molecule dimerization, it should be taken into consider-

BIOCHEMISTRY (Moscow) Vol. 83 Suppl. 1 2018 S158 SELIVANOVA et al. ation that some of these residues are responsible for the 14. Feng, X., Tang, H., Leng, J., and Jiang, Q. (2014) molecule binding to the IR. Suppressors of cytokine signaling (SOCS) and type 2 dia- 3. Since the monomeric form of insulin is more betes, Mol. Biol. Rep., 41, 2265-2274. prone to fibrillation, insulin mutants should be studied for 15. Cheng, K., Andrikopoulos, S., and Gunton, J. E. (2013) First phase insulin secretion and type 2 diabetes, Curr. Mol. their propensity for fibril formation. Med., 13, 126-139. In general, the design of receptor-selective insulin 16. Zaykov, A. N., Mayer, J. P., and DiMarchi, R. D. (2016) analogs with decreased propensity for amyloidogenesis is Pursuit of a perfect insulin, Nat. Rev. Drug Discov., 15, 425- of great importance, because it will allow the mainte- 439. nance of normal glucose blood levels without adverse side 17. Home, P., Riddle, M., Cefalu, W. T., Bailey, C. J., Bretzel, effects associated with the mitogenic action and fibrilla- R. G., Del Prato, S., Leroith, D., Schernthaner, G., Van tion of insulin preparations. Gaal, L., and Raz, I. (2014) Insulin therapy in people with type 2 diabetes: opportunities and challenges? Diabetes Care, 37, 1499-1508. 18. Dzhavakhishvili, T. S., Romantsova, T. I., and Roik, O. V. Acknowledgments (2010) Dynamics of body weight in patients with type 2 diabetes during the first year of insulin therapy, Obes. Metab., The study was supported by the Molecular and Cell 4, 13-19. Biology Program (01201353567). 19. Dedov, I. I., Shestakova, M. V., and Moiseev, S. V. (2005) Analogues of insulin, Klin. Farmakol. Ter., 14, 49-55. 20. Vigneri, R., Squatrito, S., and Sciacca, L. (2010) Insulin REFERENCES and its analogs: actions via insulin and IGF receptors, Acta Diabetol., 47, 271-278. 1. Banting, F. G., and Best, C. J. (1922) The internal secretion 21. Bell, G. I., Pictet, R. L., Rutter, W. 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